Stable immunogenic protein having multiple cysteines molecules process therefor and composition thereof

Abstract

The invention describes a stable immunogenic protein having multiple cysteines molecules wherein the protein is having stability up to two years and purity more than 98% particularly rPvRII and/or rPfF2. It also discloses a method for producing said immunogenic protein comprising the following steps: culturing the host E. coli cells containing a desired recombinant gene construct comprising a codon optimized gene sequence of rPvRII and/or rPfF2 to produce cells in high density; inducing expression rPvRII and/or rPfF2 as inclusion bodies; harvesting the cells and isolating the said inclusion bodies; separating rPvRII and/or rPfF2 from inclusion bodies by repeated sequential washing and solubilizing with chaotrophic agents comprising guanidine hydrochloride and/or urea; purifying the protein by subjecting to metal-chelate affinity chromatography; re-folding of the purified rPvRII and/or rPfF2 obtained in step e) with a redox system to recover a high yield of the soluble protein, followed by further purifying the desired protein by removing impurities by subjecting to chromatography. Further the invention discloses formulation comprising rPvRII or rPfF2, preferably being lyophilized using polysaccharides preferably sucrose, lactose, and pharmaceutically acceptable adjuvants such as aluminum hydroxide, aluminum phosphate, CpG nucleotides, non-CpG nucleotides, Montanide ISA-720, MF-59, Mono-phosphoryl Lipid-A (MPL-A) and QS-21.

1 . A stable immunogenic protein having multiple cysteines molecules wherein the protein is having stability up to two years and purity more than 98% when prepared by the process comprising:
 a) culturing the host  E. coli  cells containing a desired recombinant gene construct comprising a codon optimized gene sequence of a protein to produce cells in high density,   b) inducing expression of the target protein as inclusion bodies,   c) harvesting the cells and isolating the said inclusion bodies,   d) separating the protein from inclusion bodies by repeated sequential washing and solubilizing with chaotrophic agents comprising guanidine hydrochloride and/or urea,   e) purifying the protein by subjecting to metal-chelate affinity chromatography,   f) re-folding of the purified protein obtained in step e) with a redox system to recover a high yield of the soluble protein, followed by   g) further purifying the desired protein by removing impurities by subjecting to chromatography.   
     
     
         2 . A protein as claimed in  claim 1  is stable for twenty four months at a temperature between 2-8° C. and six months at a room temperature. 
     
     
         3 . A protein as claimed in  claim 1  is rPvRII and/or rPfF2. 
     
     
         4 . A method as claimed in  claim 1 , wherein the repeated sequential washing is effected by washing with buffer. 
     
     
         5 . A method as claimed in  claim 1 , wherein the buffer used is a low salt buffer consisting of 5-50 mM Tris, 2-25 mM EDTA, 50-100 mM sodium chloride, 1-6M urea, 0.1-1% Triton X-100, pH 6.5-7.5 followed by high salt buffer containing 5-50 mM Tris, pH 6.5-7.5 and 0.5-1M sodium chloride. 
     
     
         6 . A method as claimed in  claim 1  wherein the harvesting, separating, isolating and solubilizing is carried out under denaturing conditions. 
     
     
         7 . A method as claimed in  claim 1 , wherein the refolding is effected employing a buffer containing a detergent-dextrin mixture, a co-solvent and a redox pair. 
     
     
         8 . A method as claimed in  7 , wherein the detergent used is a non-ionic one, preferably Triton X-100 at a concentration of 0.1-1.0 mM, a dextrin, preferably beta-cyclodextrin, L-Arginine as a co-solvent and a redox pair comprising at least one of the following: oxidized and reduced glutathione; L-cysteine-cystamine dihydrochloride, preferably L-cysteine-cystamine dihydrochloride. 
     
     
         9 . A method as claimed in  7 , wherein the refolding buffer further comprising one or more of the amino acids selected from L-cystine, L-cysteine, L-proline and L-lysine. 
     
     
         10 . A method as claimed in  claim 1 , wherein the re-folded PvRII is further purified by column chromatography to be free from chaotrophic agent, detergents, monomers and multimers. 
     
     
         11 . A stable composition comprising of a protein as claimed in  claim 1  wherein the protein is present in the range of 10 μg to 100 μg and pharmaceutically acceptable adjuvants. 
     
     
         12 . A pharmaceutical composition of a protein as claimed in  claim 1  where in rPvRII or rPfF2, preferably being lyophilized using at least one of the following: sucrose, lactose, maltose, trehalose, melibiose, mannitol, sorbitol and dextrose, or combinations thereof and in the range of 5 to 10% preferably sucrose or lactose and pharmaceutically acceptable adjuvants. 
     
     
         13 . A pharmaceutical composition as claimed in  claim 12 , wherein the pharmaceutically acceptable adjuvants used are selected from, but not limited to: aluminum hydroxide, aluminum phosphate, CpG nucleotides, non-CpG nucleotides, Montanide ISA-720, MF-59, Mono-phosphoryl Lipid-A (MPL-A) and QS-21, used either singly or in combinations thereof for use as a vaccine against malarial infections. 
     
     
         14 . A pharmaceutical composition of a protein as claimed in  claim 1  is useful as vaccine for immunization by at least one of the following routes of administration: subcutaneous, intra-dermal or parental.